NIH Director’s Pioneer Award

2007 Pioneer Award Recipient Abstracts

Emotional states are central to mental and physical health. NIH invests tremendous resources in research on emotion, much of it devoted to animal models. Ironically, this research is guided by a scientific paradigm that is grounded in human experience. People experience fear and see it in others, so scientists assume there must be a literal (modular) neural circuit for fear in the mammalian brain. Rats freeze when they hear a tone paired with a foot shock, so they are presumed to be in a state of fear (versus surprise, anger, or even a general state of alarm) and undergoing “fear learning.” Scientists also presume that a map of the neural circuitry of freezing behavior will yield a neural mechanism for fear that is largely preserved in humans, and a decade of neuroimaging studies have focused on locating a homologous neural circuit in the human brain. In the last five years, I have traced the roots of this “natural kind” model, conducted a comprehensive review of the literature to examine its veracity, and found it wanting (Barrett, 2006a).1 In response, I have fashioned a new systems-level model, called the Conceptual Act Model, grounded in the neuroanatomy of the human brain. My model parsimoniously incorporates neuroscience findings from rats, primates, and humans, and explains the mechanisms that produce the range and variety of behavioral and introspective instances that we call “emotion” (Barrett, b, c; Barrett, Mesquita, Ochsner, & Gross, 2007; Barrett, Ochsner, & Gross, 2007; Duncan & Barrett, 2007). The Conceptual Act Model asks different – and perhaps better – questions about what emotions are and how they function in mental and physical health. The NIH Director’s Pioneer Award will allow me the intellectual freedom and resources to continue building evidence for the Conceptual Act Model of emotion, thereby shaping a new paradigm to guide the scientific study of emotion.

This project is designed to understand the rapid increase in autism prevalence in the United States over the past two decades. Proposed are new analyses of complex, multilevel temporally sensitive data sets that will enable me to determine the extent to which familial, environmental, gene-environment, and diagnostic drift/substitution are driving the autism epidemic. Detailed attention to and models capable of capturing social network and social influence underpinnings of the epidemic are developed. New analysis models for intercalating spatial and social network data are developed. These models where appropriate are extended to a wide range of developmental disorders that have increased rapidly in prevalence.

General anesthesia is a drug-induced, reversible condition comprised of five behavioral states: hypnosis (loss of consciousness), amnesia (loss of memory), analgesia (loss of pain sensation), akinesia (immobility), and hemodynamic stability with control of the stress response. The mechanisms by which anesthetic drugs induce the state of general anesthesia remain one of the biggest mysteries of modern medicine. Study of the neural circuitry responsible for each of the five behavioral states of general anesthesia is a fundamental question being investigated in systems neuroscience but not for the purpose of understanding anesthesia. I propose to use systems neuroscience paradigms to establish an interdisciplinary program to solve the mystery of general anesthesia. The program will consist of a set of coordinated studies in humans, monkeys and rats using the same anesthetic agents, in addition to use of dynamical systems modeling studies of anesthetic effects on neural circuits and the development of new signal processing algorithms to track in real-time the dynamics of brain states under general anesthesia. The animal studies will use established behavioral paradigms, fMRI, and multielectrode methods to track brain activity, and microinjection and novel nanoparticle methods for site-specific delivery of anesthetic drugs. The human studies will track brain states under anesthesia using fMRI and simultaneously recorded EEG. The investigators will collaborate to integrate this information across the different systems and scales. This project will lead to a more precise, neurophysiologically-based understanding of general anesthesia, safer protocols for anesthetic drug-development, site-specific methods for anesthetic drug delivery and the design of better, neurophysiologically-based methods for measuring depth of anesthesia. Therefore, this research will improve human health by reducing the risk of anesthesia-related morbidity for patients whose surgical or medical therapies require general anesthesia. This research will also have broad impact on the training of anesthesiologists by placing greater emphasis on systems neuroscience.

How do neural circuits guide our behavior? The answers promise to revolutionize our understanding of what it means to be human and how to repair the damaged neural circuits that underlie human neurological and psychiatric disorders. The incredible complexity of the mammalian brain, however, coupled with limited ability to genetically manipulate specific neural circuits in vertebrates, has made our progress difficult. My lab is developing new approaches that will rejuvenate this effort. We take advantage of the fact that many basic neural computations are evolutionarily ancient: invertebrates are capable of some of the same computations that humans are. This enables us to study processes familiar to vertebrate physiologists using the fruit fly, an animal with a relatively simple, genetically hard-wired nervous system. As a model genetic system, Drosophila offers a complex; interesting behavioral repertoire combined with an extensive toolkit for both forward and reverse genetic analysis. Our goal is to provide a complete mechanistic understanding of how visual information is processed at the level of identified cells and circuits. In preliminary work, we have developed new behavioral paradigms that allow high-throughput, automated forward genetic screens to identify neurons specifically involved in such processes as motion detection and color perception. To define the behavioral contributions of these functionally important neurons, we are adapting analytical techniques from ion channel biophysics and systems neuroscience to the analysis of fly behavior. Using new molecular and electrophysiological techniques that we will develop, we propose to link circuit anatomy to circuit function, and to define how changes in the activities of functionally important neurons lead to behavioral decisions. These studies will provide the first synthesis linking a sensory input to a behavioral output, through the functions of specific molecules, neurons and circuits.

The goal of this project is to use innovative systems biology and synthetic biology approaches to quantitatively characterize and analyze bacterial gene regulatory networks underlying cellular responses to antibiotics, the formation of persisters and the emergence of resistance. With the alarming spread of antibiotic-resistant strains of bacteria, a better understanding of the specific sequences of events leading to cell death from bactericidal antibiotics is needed for future antibacterial drug development. Accordingly, there is a need for systems biology and synthetic biology approaches to discern the interplay between genes, proteins and pathways in furthering our understanding of how bacteria respond and defend themselves against antibiotics. The implications of the underlying logic of genetic networks are difficult to deduce through experimental techniques alone, and successful approaches will in many cases, involve the union of new experiments and computational modeling techniques. To address this problem, we have developed computational-experimental methods that enable construction of quantitative models of gene, protein and metabolite regulatory networks using expression measurements and no prior information on the network structure or function. In this project, we will use these approaches to reverse engineer bacterial gene regulatory networks underlying cellular responses to antibiotics, the formation of persisters and the emergence of resistance. The resulting networks and pathways will be analyzed to gain insight into the regulatory control of the associated biological processes, and the network models will be used to identify key regulators and mediators for a variety of phenotypic responses. This work could lead to new insights into the stress response of bacteria and the identification of novel targets for drug discovery, e.g., ones that overcome bacterial protective mechanisms or activate bacterial programmed cell death. This project may thus enable the development of novel classes of antibiotics that account for and utilize the complex regulatory properties of genetic networks.

A major challenge in cell and organism biology is to understand how living cell physiology emerges from the biophysical properties of individual macromolecules. The morphological and physical behaviors of cells required for cell adhesion, migration and division depend on the proper spatial and temporal regulation of a vast hierarchy of multi-protein machines, called the cytoskeleton. However, while we are gaining increasing amounts of knowledge of properties of individual cytoskeletal proteins, we have very little knowledge about the self-assembly and physical properties of multi-protein assemblies that form physical structures to transmit mechanical information up to cellular length scales. For example, we do not understand how forces generated by individual molecular motors are exploited by cytoskeletal assemblies to regulate morphogenesis and force generation at the cellular level. Current understanding of the physical behavior of the cellular cytoskeleton has been limited both by the lack of experimental techniques to probe the dynamic structure and physical properties of mesoscopic cytoskeletal assemblies in living cells. I propose to establish the experimental tools to study the biophysical properties of cytoskeletal matter in living cells by integrating approaches from condensed matter physics with molecular cell biology. This work will identify the underlying physics of emergent cytoskeletal assemblies and will provide predictive analytical models to link our understanding of the biophysics of molecules to cell behaviors. Finally, this work will impact the treatment of diseases that are a result of misregulation of the physical behaviors of cells, including cancer metastasis and cardiac diseases.

How does early experience shape ourselves? We have shown such critical period brain development is triggered by specific parvalbumin (PV)-positive GABA cells, then hard-wired by sequential re-configuration of spines and inputs upon pyramidal cell dendrites. At a genomic level, a far more widespread world of non-coding RNA (ncRNA) has been identified than previously anticipated. Accelerated regions of change in ncRNA sequences expressed in strategic cell types appear vital to human brain evolution. By rapidly regulating mRNA translation even distally, ncRNAs may be particularly adapted to respond to constantly changing environments, defining a unique milieu for maintaining the identity of individual neurons. Strikingly, the role of ncRNAs in brain function (and dysfunction) remains virtually unknown. We will explore their contribution to the onset and permanence of critical period brain development. Using replication-defective adenoviral vectors, we will develop “pulse gene transfer” into specific progenitor cells in a neuronal birthdate-specific manner in mice. By covalently linking magnetic beads, innovative constructs containing specific ncRNA sequences or inducible Crerecombinases will be focused in utero for late over-expression or deletion of endogenous ncRNAs in PV-cells. Virally infected pyramidal cell cohorts will similarly be manipulated postnatally by their canonical inside-out laminar origins. An integrated assessment of electrophysiological, optical imaging, anatomical and behavioral measures of vision, audition and social behaviors will be performed on these various mouse models. We will then test the hypothesis that ncRNAs act as molecular switches to regulate gene networks within neural networks. By coordinating maturation of PV-cells and the propagation of well-orchestrated changes across cortical layers, ncRNA may establish the timecourse of experience-dependent brain plasticity. Behavioral and physiological reactivation in adulthood would elucidate the purpose of critical periods. Importantly, our work will identify novel methods and therapeutic targets for developmental brain disorders, like autism or schizophrenia, which tragically incarcerate the mind.

Cell fate maps describe how the sequence of cell division, migration, and apoptosis transform a zygote into an adult and are fundamental to understanding stem cell biology. Yet, it is only in the transparent worm C. Elegans where tedious microscopic observation of each cell division has allowed for construction of a complete cell fate map. More complex—and opaque—animals prove less yielding. DNA replication, however, inevitably generates somatic mutations. Consequently, multicellular organisms comprise mosaics where most cells acquire unique genomes that are potentially capable of delineating their ancestry. We propose to construct mammalian cell fate maps using a phylogenetic approach to passively retrace embryonic relationships by deducing the order in which mutations have arisen during development. We have found that polyguanine repeat DNA sequences are particularly useful genetic markers, because they frequently change length during mitosis. To demonstrate feasibility, we have used phylogenetics to reconstruct the lineage of cultured mouse NIH3T3 fibroblasts based on mutations affecting the length of polyguanine markers. We have then employed whole genome amplification to genotype polyguanine markers in single cells taken from a mouse and used phylogenetics to infer the developmental relationships of the sampled tissues. Our preliminary results demonstrate the potential of this approach for retrospectively producing a complete mammalian cell fate that, in principle, could describe the developmental lineage of any cell and resolve outstanding questions relevant to stem cell biology.

This proposal describes a multi-disciplinary research program that aims to develop, validate, and disseminate microfluidic technologies for quantitative studies of protein aggregation and aging. Protein aggregation is associated with aging and with a number of human diseases that affect both quality and duration of life. Many fundamental aspects of protein aggregation remain elusive, including connections between protein aggregation and toxicity, and the connection between protein aggregation and initiation and progression of diseases. Microfluidic platforms will be developed to understand these complex processes from both bottom-up and top-down perspectives. Bottom-up, new droplet-based microfluidic systems will be developed to characterize quantitatively the connection between protein aggregation and toxicity in vitro. This system will allow the reproducible real-time generation, manipulation, and characterization of aggregates for in vitro and in vivo toxicity screens. Multidimensional statistical analysis of toxicity patterns obtained in these devices may elucidate the connection between protein aggregation and toxicity, clarify the mechanism of action of existing drug candidates that target aggregation, and accelerate development of new drugs and drug cocktails. Top-down, microfluidic technologies will be developed to induce and monitor aggregation in vivo with high spatiotemporal resolution, and to observe the effects of aging, physiological state, neuronal activity, and presence of drug candidates on the initiation and progression of protein aggregation diseases. These two technologies will be used together to understand protein aggregation and aging, and may lead to new hypothesis and molecules for controlling these processes.

Epilepsy is a disorder that involves far more than the occurrence of seizures, and seizures can cause neuronal network disturbances that result in a wide range of cognitive and behavioral impairment. To date, most work in the epilepsy field has centered on the mechanism or prevention of the ictal events themselves. The focus of my laboratory has been on the impact of early life seizures on brain development and epileptogenesis. The present proposal extends our work to determine whether these mechanisms also induce alterations that could lead to cognitive dysfunction manifesting in early life, such as autism. There is clinical evidence that early life seizures may be one of many precedents for autism, and epilepsy is common in patients with autism, suggesting an interaction between the two processes. Our prior and recent work suggests that at least in the immature brain, where baseline synaptic plasticity is enhanced, seizures appear to directly activate specific plasticity-associated signaling pathways. We hypothesize that seizure induced “dysplasticity” may occlude normal plasticity involved in cognition, and induce abnormal patterns of synapse development similar to those observed in autism and other forms of neurodevelopmental delay. Using electrophysiological techniques, we will first examine the time course of seizure-induced interruption of normal synaptic plasticity in the immature brain. We will then determine whether specific activity-dependent signaling abnormalities known to be associated with autism occur de novo following seizures in the immature brain. Next, we will identify seizure induced mechanisms for their activation and test whether post-seizure intervention attenuates the altered structure and function of neuronal networks. Finally, we will determine whether similar alterations in signaling, regulatory, and synaptic proteins also are observed in human tissue following seizures and in cases of autism associated with neonatal or infantile seizures.

I propose a disruptive technology that will revolutionize our understanding of brain function, development, and disease. Because the study of neural circuits remains deeply limited by a paucity of data, we need massively parallel approaches to brain imaging that will raise data acquisition rates by over two orders of magnitude. High-throughput technologies have already revolutionized certain areas of biology such as genomics and proteomics, but neuroscience has yet to experience a growth spurt of comparable magnitude. I will construct instrumentation allowing the brain volumes of ~100 alert flies to be imaged simultaneously by two-photon fluorescence microscopy. I have chosen the fruit fly, Drosophila melanogaster, because of its small brain, its sophisticated behavioral repertoire, the large number of strains with genetically targeted alterations to brain circuitry, the utility of fluorescence imaging of neural activity in this species, and the importance of the fly as a model for the study of many brain diseases. Massively parallel brain imaging will open entirely new avenues: 1) The ability to track neural dynamics across the brains of large numbers of normal flies and those with genetically induced neural circuit perturbations will revolutionize our understanding of how neural circuits produce animal behavior; 2) The now prominent role of the fruit fly as a model system for the study of developmental disorders, neurodegenerative diseases, and addiction implies we will gain significant medical insights into devastating conditions; 3) Our technology will have important applications to drug screening, allowing the cellular effects of new compounds to be assessed rapidly in vivo; 4) The ability to perform high-throughput time-lapse imaging of cellular events during the maturation of fly embryos will allow an additional revolution in developmental neurobiology. Applications of our technology will also be plentiful in other model organisms such as nematodes and zebrafish, profoundly impacting multiple areas of biomedicine.

Memory and other cognitive functions reside in part in the pattern and strength of synaptic connections between neurons. Understanding the molecular determinants of synaptic strength has been a longstanding goal of neuroscience, and advances in this field stand to influence our understanding of virtually every neurological disorder from Autism to Alzheimer’s disease. Over the past decade biochemical and conventional molecular and genetic approaches have begun to piece together how interactions between neurotransmitter receptors and other synaptic proteins regulate and control synaptic strength and plasticity, but a major limitation is that there is little or no structural information about how proteins are arranged into signaling complexes at the synapse. Many signaling molecules can only interact with immediately adjacent proteins, and this localization may itself be regulated by experience. Understanding how functional signaling complexes are generated and how they in turn regulate synaptic strength thus requires that we probe the spatial arrangements of proteins within the postsynaptic density (PSD). Conventional approaches do not have sufficient resolution to allow the position of synaptic proteins to be mapped within these tiny (< 1 μm) synaptic structures. Here I propose to develop tools to map the spatial arrangements of individual synaptic proteins (such as glutamate receptors) within the PSD, and to determine how these spatial arrangements are influenced by synaptic plasticity, using super resolution light microscopy. By mapping the relative positions of many different proteins within the postsynaptic membrane and PSD we will be able to generate a 3 dimensional model of the protein lattices that comprise the postsynaptic side of the synapse. This method has the promise to put a vast array of biochemical and molecular data on protein-protein interactions into a structural context that is essential for its interpretation, and will add a powerful new tool to the analysis of synaptic function.

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